A tentative schedule of things we will do over the first few weeks.
Sadhana, Joshua, Hong, Jeanette, Marshall, Justin, Larry, Joey, Krishna, Christina, J.C., Indra, Benjamin, Vanna, Keyur, Omkar, Sonny

Research Presentation Schedule:
July 12th: Joey, Indra, Christina
July 19th: Christina, J.C., Sadhana, Keyur, Larry
July 26th: Krishna, Jeanette, , Marshall
August 2nd: Justin, Hong, Ben, Sonny, Joshua
August 9th:

Instructions: prepare 3-5 Powerpoint slides to present your work to date
  • Intro (talk about your disease and what your protein does), Objectives, Methods& Results, Future Directions


NOTE (as of 07/19/11): use new grant account number for DNA Sequencing jobs (it is already on the form for you in a pull-down list)- otherwise get it from Dr. B or Thao.



Friday Aug 12th

10:30 - start prepping plate 1 for RT-PCR
12:00 - 2:00 RT-PCR run on 7900-A machine

Set up plate 2 afterwards and run on 7500 machine















































MONDAY - June 6th
4 plates of LB with Amp
Make 250 ml of LB
Nanodrop plasmid DNA
submit plasmid to DNA sequencing facility (pGFP or pmCherry)


TUESDAY - 7th
12:00 Lunch meeting

Make Amp and Kan stocks (50ug/ul)
4 people: Transform a plasmid for Midi-prep (pGBR22)
rest: Transformation Efficiency

Paper copy of safety training to Dr. B

WEDNESDAY - 8th
10:30 - Life Technologies talk - Nitin Puri - students invited (in Bio 301) - VDSers meet in PAI 2.14 lab 10 minutes beforehand

Check DNA sequence
Analyze DNA sequence exercise - in computer lab

5:00 - Krishna and Christina - pick colony of pGBR22 to grow up overnight

THURSDAY - 9th
11:00 - Krishna and Christina - spin down and then Midi-prep pGBR22
RE digest and agarose gel check
Dilute some primers

Print out safety training, hole punch and place in VDS sign in folder under the 'Safety' tab.


FRIDAY - 10th
OFF - midiprep?

MONDAY - June 13th
OFF



TUESDAY - June 14th

HSers -
  • lab safety training and printout
  • Micropipettor calibration
  • Nanodrop, submit DNA to sequencing


Transformation of plasmids to make stocks - pUC19, pNIC-Bsa4 (Sadhana, Jeanette, Marshall, Justin)
Dilute some primers (Joey, Larry) - see PrimerDilution instructions on GDocs in MISC collection
Make Ampicillin and Kanamycin stocks (J.C., Indra)
Catch-up (Hong and Joshua)

Krishna, Christina - show Ben, Vanna, Keyur, and Omkar how to do Micropipette calibration, how to do Nanorop and DNA sequencing


Lab Notebook check

4:00 Lab Group Meeting

WEDNESDAY - June 15th

HSers -
  • lab safety training and printout
  • RE digest & Agarose Gel
  • Transformation
  • Make LB and plates (if not already)

Target Selections

Grow up plasmids overnight - pUC19 Sadhana, Jeanette


THURSDAY - June 16th


11:00 - Midi-prep pUC19 - Sadhana, Jeanette

HSers -
  • lab safety training and printout
  • Anaylyze DNA Sequence
  • Transformation
  • Make LB and plates (if not already)


PCR of pGBR22
Agarose Gel of PCR



Transform pNIC-Bsa4 (Marshall, Justin)


FRIDAY - June 17th


1:00 - Meet in computer lab to do Primer Design

ProtocolPCRforREcloningGREEN.xls OR ProtocolPCRforREcloningRED.xls
ProtocolPCR_pLICsequencing
ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Fall10.doc


HSers: lab safety training, RE Digest?, PCR


Monday - June 20th
ProtocolPCRforREcloningGREEN.xls OR ProtocolPCRforREcloningRED.xls
ProtocolPCR_pLICsequencing
ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Fall10.doc
Grow up plasmids overnight pNIC-Bsa4 (Marshall, Justin, Hong, Indra)
HSers - PCR, PyMol intro to CA2?

Tuesday - June 21st
10:30 - Marshall, Indra Spin down and MidiPrep pNIC-Bsa4
Update individual Wikispaces pages
Create a Wikispaces page specifically for your target (if you are working in pairs - only create one page and split up the work) . Post relevant target info there - see examples already on Targets page.
Christina - check in PTP1b culture grew successfully overnight. If not - then do transformation.
Midi-prep pUC19 pNIC-Bsa4 (Marshall, Justin)
ProtocolPCR_pLICsequencing
ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Summ11.doc

1:30 - HSers - Analyze DNA sequence exercise

4:00 Group Meeting
  • Journal Club
Lab Notebook check -


Lab notebook check - moved to tomorrow

Joey, JC - overnight growth of pUC19 in DHFalphas
Indra, Hong - overnight growth of pNIC-Bsa4
Indra, Hong - Transformation of PTP1b in pNIC-Bsa4 in DH5alphas


Wednesday - June 22nd
10:15 - Midiprep (Joey, JC, Indra) - spin down and save for after Fire Safety Training
11:00 - Fire Safety training
2-4 pm Middle School students for lab demo (2)
Sadhana, Larry - Make 1 Molar IPTG, sterile filter, store in -20 degC (make 20 tubes of 500 ul each) - label "1M IPTG" with date
Autoclave 4 x 2L flasks for expression
5:00 - Lab Group Meeting Follow up ( cover slides we didn't get to yesterday)
6:00 - Christina, Sadhana, Christina, Jeanette, Marshall pick colony of PTP1b in pNIC-Bsa4 for o/n expression in BL21(DE3) cells in 50 ml of LB
Lab Notebook check
Larry, Joshua -overnight growth of PTP1b in pNIC-Bsa4 in DH5alphas (from Indra and Hong's transformation)

Thursday - June 23rd
10:00 Protein Expression of PTP1b (4 people) - Sadhana, Christina, Jeanette, Marshall


~ 5:00 Joshua, Larry - grow up plasmid in bacteria overnight for Midi-prep tomorrow



Friday - June 24th
11:00 Joshua, Larry, Hong - Midiprep of PTP1b in pNIC-Bsa4 in DH5alphas Hong midiprep of pNIC-Bsa4 in DH5alphas

11:00 - Sadhana, Jeanette, Marshall, Christina - sonicate, etc on protein samples

HSers - lab notebook check

Krishna out
Ben - off for 3 weeks



Monday - June 27th
1:00 - Sadhana, Jeanette, Marshall, Christina - Protein Purification &

Continue PCRs from last week

Primer design for pNIC-Bsa4 cloning strategy primers with Lagger and Leader
- use these:

Start Primer Overlap PCR - protocol on GDocs
ProtocolPCR_PrimerOverlapVDS_Summ11.docx

Tuesday - June 28th

11:00 - Christina, Josh - make and run SDS-Page protein gels
11:30 - Jeanette and Sadhana - load and run SDS-Page protein gels

EVERYONE - do Primer Design for pNIC-Bsa4 cloning strategy (see Monday's list)


4:00 Group Meeting
  • Journal Club
  • Lab Notebook Check

After Class: Joey, J.C., Larry, Joshua - prepare LB, flasks and grow up overnight culture of protein



Wednesday - June 29th
10:30 (all day) - Express protein: Joey, J.C., Larry, Joshua
EVERYONE - do Primer Design for pNIC-Bsa4 cloning strategy (see Monday's list)

Start cloning of PCR from Primer Overlap into pUC19
  • ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc



Thursday -June 30th
12:00 - Lyse and spin down protein: Joey, J.C., Larry, Joshua

After you have done Overlap PCRs - you can proceed to clonig (be sure to read how long the steps will take so that you can plan it out!)
ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc

Jeanette, Sadhana, Christina, Joshua - run FPLC on concentrated sample


Friday - July 1st
11:00 - Purify protein: Joey, J.C., Larry, Joshua (and make gels for tomorrow)


That's right - it's time to challenge your lab mates to a friendly game of laser tag! All PAI 2.14 researchers & anyone else who wants to go are invited! Here are the details:

Friday, July 1st 2:30 pmhttp://www.blazertag.com/









Price: $7.25 per person per game for a group bigger then 10 Phone: 512-462-0202









1701 W. Ben White BLVD. Austin TX 78704

*We'll meet in PAI 2.14 at 2:30 & leave shortly after that (~2:40). We can coordinate carpools at that time.*



Monday July 4th - OFF

Tuesday July 5th
If you have finished Secondary PCR, then Start cloning (take approximately 6-7 hrs to do)
The first 3 cloning steps need to be done on the same day (Restriction Digest, Ligation, Transformation)
Options:
A. Start early and finish before class at 4:00
B. Start as usual but time it so that the ligation step (1-2 hrs) is during the class time.

ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc

Joey, J.C., Larry, Marshall - concentrate protein sample and make protein gels

4:00 - Lab Group meeting



Wednesday July 6th


2:00 Joey, J.C., Larry, Marshall - run protein samples on GEL

If you did not get colonies on your plate, you can start working on the Version 2 of the pUC19 Cloning strategy
ProtocolCloningLigate_REdigest_Transform_pUC19_VDS_Summer11_version2.doc



Thursday July 7th
The new Journal Club paper is uploaded HerschhornHIVreversetranscriptaseVirtualScreenJMedChem2008.pdf
Joey, J.C., Larry, Marshall - run FPLC on samples


Friday July 8th
Dr. B out - work on cloning

Turn in Lab Notebook to 'Drug Screening Notebooks' drawer in Painer 2.14 Lab - for check by Dr. B

Day 1:
  • Digest, ligate, transform (long day)
Day 2:
  • Grow up colonies
  • ------------------------------ (simultaneously start alternative strategy)
Day 3:
  • Mini prep colonies and send to sequencing
Day 4:
  • Wait
Day 5:
  • Get sequencing results



Monday July 11th

Need lab notebooks from: Christina, Larry, Justin, Joshua

Update your Wikispaces research page to show latest gel images.

Verify genes within pNIC-Bsa4 by checking your DNA sequencing results of pLIC primers on pNIC-Bsa4


afternoon (~1 ish)
Joey, J.C., Larry, Marshall - Test (the other guys') PTP1b in enzyme test
Jeanette, Sadhana, Christina, Joshua - Test your PTP1b in enzyme test
See protocol on Google Docs

5:00 - Justin, Indra, Hong, Krishna - grow PTP1b protein overnight in small flasks



Tuesday July 12th
10:00 -Justin, Indra, Hong, Krishna - express PTP1b protein

4:00 Group Meeting and Journal Club (Jeanette's TGF-Beta paper) + Larry
Research Presentation: Joey & Indra
The new Journal Club paper is uploaded HerschhornHIVreversetranscriptaseVirtualScreenJMedChem2008.pdf


Wednesday July 13th
Justin, Indra, Hong, Krishna - work on PTP1b protein sonication, purification

Thursday July 14th
Justin, Indra, Hong, Krishna - work on PTP1b protein making gels, running gels

Friday July 15th
Justin, Indra, Hong, Krishna - work on PTP1b protein FPLC purification
Day 1:
  • Digest, ligate, transform (long day)
Day 2:
  • Grow up colonies
  • ------------------------------ (simultaneously start alternative strategy)
Day 3:
  • Mini prep colonies and send to sequencing
Day 4:
  • Wait
Day 5:
  • Get sequencing results



Monday July 11th

Need lab notebooks from: Christina, Larry, Justin, Joshua

Update your Wikispaces research page to show latest gel images.

Verify genes within pNIC-Bsa4 by checking your DNA sequencing results of pLIC primers on pNIC-Bsa4


afternoon (~1 ish)
Joey, J.C., Larry, Marshall - Test (the other guys') PTP1b in enzyme test
Jeanette, Sadhana, Christina, Joshua - Test your PTP1b in enzyme test
See protocol on Google Docs

5:00 - Justin, Indra, Hong, Krishna - grow PTP1b protein overnight in small flasks



Tuesday July 12th
10:00 -Justin, Indra, Hong, Krishna - express PTP1b protein

4:00 Group Meeting and Journal Club (Jeanette's TGF-Beta paper) + Larry
Research Presentation: Joey & Indra
The new Journal Club paper is uploaded HerschhornHIVreversetranscriptaseVirtualScreenJMedChem2008.pdf


Wednesday July 13th
Justin, Indra, Hong, Krishna - work on PTP1b protein sonication, purification

Thursday July 14th
Justin, Indra, Hong, Krishna - work on PTP1b protein making gels, running gels

Friday July 15th
Justin, Indra, Hong, Krishna - work on PTP1b protein FPLC purification



Monday July 18th

Need lab notebooks for last time from: Larry, Justin, Joshua
Update your Wikispaces research page to show latest gel images.

Justin, Indra, Hong, Krishna - work on PTP1b protein: make gels, run gels, stain and then destain overnight, concentrate protein,

Thao - prep 2x Restriction Enzyme digest of pGBR22 for Middle Schoolers on Wednesday

Tuesday July 19th

  • reconcentrate protein after gel

4:00 Group Meeting and Journal Club Hong & Indra (paper copies of the journal articles are in the lab - in the Drug Screening Notebooks drawer)
Research Presentation: J.C., Sadhana, Keyur



Wednesday July 20th


2-4 pm: Middle Schoolers 'shadowing' in lab

Journal Club paper for Joshua is uploaded (paper copies of the journal articles are in the lab - in the Drug Screening Notebooks drawer)


Thursday July 21st
10:00 -5:00 - RT-qPCR workshop: 1st Group: Sadhana, Jeanette, Joey, Krishna, J.C., Larry
  • Next week: 2nd Group: Justin, Joshua, Hong, Marshall, Indra
Justin, Indra, Hong, Krishna - run PTP1b protein on FPLC with Dr. B and Thao


Friday July 22nd

1:30 - Analyze Data from RT-qPCR workshop: 1st Group: Sadhana, Jeanette, Joey, Krishna, J.C., Larry

~1:00 Justin, Indra, Hong, Krishna - run PTP1b protein on FPLC with Dr. B and Thao

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Monday July 26th

10:00 am -6:00 pm - RT-qPCR workshop:n 2nd Group: Justin, Joshua, Hong, Marshall, Indra ...... (the new cDNA came in - so we are on for Monday)

Justin, Indra, Hong, Krishna - PTP1b enzyme activity test

???Thao - prep 2x Restriction Enzyme digest of pGBR22 for Middle Schoolers on Wednesday

Tuesday July 27th


3:00 Justin - FPLC on PTP1b protein sample ??

4:00 Group Meeting and Journal Club - Joshua (paper copies of the journal articles are in the lab - in the Drug Screening Notebooks drawer)
Research Presentation: Krishna, Jeanette, Joshua, Marshall



Wednesday July 28th


????2-4 pm: Middle Schoolers 'shadowing' in lab



Thursday July 29th



Friday July 30th


NEXT WEEK:


Monday Aug 1st

Tuesday Aug 2nd
Thao - prep 4x Restriction Enzyme digest of pGBR22 for Middle Schoolers on Wednesday (enough for this week and next time)

4:00 Group Meeting
Research Presentation: Justin, Hong, Ben, Sonny, Joshua

Wednesday Aug 3rd

Thao - prep 2 TAE gels also
2-4 pm: Middle Schoolers 'shadowing' in lab

Thursday Aug 4th


Friday Aug 5th
Last day of summer research


NEXT WEEK: nothing ........